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Increasing the chemical space of proteins in living cells via genetic code expansion.
Krauskopf, K, Lang, K
Current opinion in chemical biology. 2020;:112-120
Abstract
In recent years it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties (dubbed unnatural amino acids, UAAs) into proteins in living cells by expanding the genetic code. Together with developments in chemistries that are amenable to and selective within physiological settings, these strategies have started to have a big impact on biological studies, as they enable exciting in cellulo applications. Here we highlight recent advances to covalently stabilize transient protein-protein interactions and capture enzyme substrate-complexes in living cells using proximity-triggered and residue-selective photo-induced crosslinking approaches. Furthermore, we describe recent efforts in controlling enzyme activity with photocaged UAAs and in extending their application to a variety of enzymatic scaffolds. In addition, we discuss the site-specific incorporation of UAAs mimicking post-translational modifications (PTMs) and approaches to generate natively-linked ubiquitin-protein conjugates to probe the role of PTMs in modulating complex cellular networks.
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2.
Reprogramming extracellular vesicles with engineered proteins.
Shi, X, Cheng, Q, Zhang, Y
Methods (San Diego, Calif.). 2020;:95-102
Abstract
Extracellular vesicles (EVs) have been emerging as a new class of cell-free therapy for the treatment of a variety of diseases, including cancer, tissue injuries, and inflammatory diseases. Reprograming native EVs by genetic engineering and other approaches offers an attractive prospect of extending therapeutic capabilities of EVs beyond their natural functions and properties. In this review article, we survey the state-of-the-art methods of EVs engineering and summarize major therapeutic applications of the reprogrammed EVs.
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3.
Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis.
Smolskaya, S, Logashina, YA, Andreev, YA
International journal of molecular sciences. 2020;(3)
Abstract
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
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4.
Practically useful protein-design methods combining phylogenetic and atomistic calculations.
Weinstein, J, Khersonsky, O, Fleishman, SJ
Current opinion in structural biology. 2020;:58-64
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Abstract
Our ability to design new or improved biomolecular activities depends on understanding the sequence-function relationships in proteins. The large size and fold complexity of most proteins, however, obscure these relationships, and protein-optimization methods continue to rely on laborious experimental iterations. Recently, a deeper understanding of the roles of stability-threshold effects and biomolecular epistasis in proteins has led to the development of hybrid methods that combine phylogenetic analysis with atomistic design calculations. These methods enable reliable and even single-step optimization of protein stability, expressibility, and activity in proteins that were considered outside the scope of computational design. Furthermore, ancestral-sequence reconstruction produces insights on missing links in the evolution of enzymes and binders that may be used in protein design. Through the combination of phylogenetic and atomistic calculations, the long-standing goal of general computational methods that can be universally applied to study and optimize proteins finally seems within reach.
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5.
Engineering Plant Cytochrome P450s for Enhanced Synthesis of Natural Products: Past Achievements and Future Perspectives.
Shang, Y, Huang, S
Plant communications. 2020;(1):100012
Abstract
Cytochrome P450s (P450s) are the most versatile catalysts and are widely used by plants to synthesize a vast array of structurally diverse specialized metabolites that not only play essential ecological roles but also constitute a valuable resource for the development of new drugs. To accelerate the metabolic engineering of these high-value metabolites, it is imperative to identify and characterize pathway P450s, and to further improve their activities through protein engineering. In this review, we focus on P450 engineering and summarize the major strategies for enhancing the stability and activity of P450s and successful cases of P450 engineering. Studies in which the functions of P450s were altered to create de novo metabolic pathways or novel compounds are discussed as well. We also overview emerging tools, specifically DNA synthesis, machine learning, and de novo protein design, as well as the evolutionary patterns of P450s unveiled from a massive number of DNA sequences that could be integrated to accelerate the engineering of these enzymes. These approaches would greatly aid in the exploitation of plant-specialized metabolites or derivatives for various uses including medical applications.
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Small design from big alignment: engineering proteins with multiple sequence alignment as the starting point.
Wang, T, Liang, C, Hou, Y, Zheng, M, Xu, H, An, Y, Xiao, S, Liu, L, Lian, S
Biotechnology letters. 2020;(8):1305-1315
Abstract
Multiple sequence alignment (MSA) is a fundamental way to gain information that cannot be obtained from the analysis of any individual sequence included in the alignment. It provides ways to investigate the relationship between sequence and function from a perspective of evolution. Thus, the MSA of proteins can be employed as a reference for protein engineering. In this paper, we reviewed the recent advances to highlight how protein engineering was benefited from the MSA of proteins. These methods include (1) engineering the thermostability or solubility of proteins by making it closer to the consensus sequence of the alignment through introducing site mutations; (2) structure-based engineering proteins with comparative modeling; (3) creating paleoenzymes featured with high thermostability and promiscuity by constructing the ancestral sequences derived from multiple sequence alignment; and (4) incorporating site-mutations targeting the evolutionarily coupled sites identified from multiple sequence alignment.
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Genetically encoded biosensors based on innovative scaffolds.
Moeyaert, B, Dedecker, P
The international journal of biochemistry & cell biology. 2020;:105761
Abstract
Genetically encoded biosensors are indispensable tools for visualizing the spatiotemporal dynamics of analytes or processes in living cells in vitro and in vivo. Their widespread adaptation has gone hand in hand with the development of sensors for new analytes or processes and improved functionality and robustness. In this review, we highlight some of the recent advances in genetically encoded biosensor development, with a special focus on novel and innovative scaffolds that will lead to new possibilities in the future.
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8.
Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.
Hatlem, D, Trunk, T, Linke, D, Leo, JC
International journal of molecular sciences. 2019;(9)
Abstract
The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.
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9.
Current Trends in Protein Engineering: Updates and Progress.
Sinha, R, Shukla, P
Current protein & peptide science. 2019;(5):398-407
Abstract
Proteins are one of the most important and resourceful biomolecules that find applications in health, industry, medicine, research, and biotechnology. Given its tremendous relevance, protein engineering has emerged as significant biotechnological intervention in this area. Strategic utilization of protein engineering methods and approaches has enabled better enzymatic properties, better stability, increased catalytic activity and most importantly, interesting and wide range applicability of proteins. In fact, the commercialization of engineered proteins have manifested in economically beneficial and viable solutions for industry and healthcare sector. Protein engineering has also evolved to become a powerful tool contributing significantly to the developments in both synthetic biology and metabolic engineering. The present review revisits the current trends in protein engineering approaches such as rational design, directed evolution, de novo design, computational approaches etc. and encompasses the recent progresses made in this field over the last few years. The review also throws light on advanced or futuristic protein engineering aspects, which are being explored for design and development of novel proteins with improved properties or advanced applications.
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10.
Near-Infrared Fluorescent Proteins and Their Applications.
Karasev, MM, Stepanenko, OV, Rumyantsev, KA, Turoverov, KK, Verkhusha, VV
Biochemistry. Biokhimiia. 2019;(Suppl 1):S32-S50
Abstract
High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.